In vitro identification of the human cytochrome P450 enzymes involved in the metabolism of R(+)- and S(-)-carvedilol.

Both the R(+) and the S(-) enantiomers of carvedilol were metabolized in human liver microsomes primarily to 4'- (4OHC) and 5'-(5OHC) hydroxyphenyl, 8-hydroxy carbazolyl (8OHC) and O-desmethyl (ODMC) derivatives. The S(-) enantiomer was metabolized faster than the R(+) enantiomer although the same P450 enzymes seemed to be involved in each case. A combination of multivariate correlation analysis, the use of selective inhibitors of P450, and microsomes from human lymphoblastoid cells expressing various human P450s enabled phenotyping of the enzymes involved in the oxidative metabolism of carvedilol. CYP2D6 was primarily responsible for 4OHC and 5OHC production, although considerable activity was observed in a CYP2D6 poor metabolizer liver and the variability of these activities across a human liver bank was not high. There was some evidence that CYP2E1, CYP2C9, and CYP3A4 were also involved in the production of these metabolites. CYP1A2 was primarily responsible for the 8OHC pathway with additional contributions from CYP3A4. The ODMC was clearly associated with CYP2C9 with some evidence for the partial involvement of CYP2D6, CYP1A2, and CYP2E1. With its complex P450 phenotype pattern and the known contribution of non-oxidative pathways of elimination, the activity (or lack of activity) of any particular P450 would have a limited influence on the disposition of carvedilol in an individual.

Species differences in the stereoselectivity of N-oxygenation of N-ethyl-N-methylaniline in vitro.

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be stereoselectively N-oxygenated in the presence of hepatic microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. In order to characterise this reaction further, the in vitro metabolism of EMA in the presence of hepatic microsomal preparations derived from a number of laboratory species has been examined. EMA N-oxide formation was stereoselective with respect to the (-)-S-enantiomer in the presence of microsomal preparations from all species examined, with the degree of selectivity decreasing in the order of rabbit > rat approximately LACA mouse approximately DBA/2Ha mouse > guinea-pig > dog. The enantiomeric composition of the metabolically derived EMA N-oxide appeared to be determined solely by the differential rate of formation of the two enantiomers as opposed to any differences in affinities for the substrate in its pro-R and pro-S conformations. The use of enzyme inhibitors, activators and inducers indicated that EMA N-oxide formation was predominantly mediated by FMO in the presence of rabbit hepatic microsomes and that these agents did not generally affect the stereochemical outcome of the biotransformation.

Species variability in the stereoselective N-oxidation of pargyline.

The monoamine oxidase inhibitor pargyline (N-benzyl-N-methyl-2-propynylamine) is known to undergo extensive in vitro microsomal N-oxidation, thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Formation of the pargyline N-oxide (PNO) metabolite creates a chiral nitrogen centre and thus asymmetric oxidation is possible. This study describes a reverse-phase high-performance liquid chromatographic (HPLC) method for the quantitation of PNO and a chiral-phase HPLC method for the determination of the enantiomeric ratio of PNO. In vitro microsomal N-oxidation of pargyline was found to be highly stereoselective in a number of species, with the (+)-enantiomer being formed preferentially. This metabolic transformation was stereospecific when purified porcine hepatic FMO was used as the enzyme source.

Asymmetric metabolic N-oxidation of N-ethyl-N-methylaniline by purified flavin-containing monooxygenase.

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be metabolically N-oxygenated in vitro with microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Microsomal N-oxygenation of EMA is known to be stereoselective and varies between species. In order to further characterise this metabolic transformation, we have examined the in vitro metabolism of EMA using purified porcine hepatic FMO. Following incubation of EMA with purified FMO, EMA N-oxide, the only metabolic detected, was found to be produced stereoselectively [ratio (-)-(S):(+)-(R), ca. 4:1]. The enantiomeric ratio of the N-oxide product did not change markedly with respect to time, enzyme or substrate concentration. Determination of the kinetics of formation of the N-oxide indicated a single affinity for the prochiral substrate with differential rates of formation of the enantiomers. The extent of EMA N-oxide formation was shown to be affected by activators and inhibitors of FMO and pH, but its stereoselectively was unaltered.

Stereoselective microsomal N-oxidation of N-ethyl-N-methylaniline.

The stereoselectivity of metabolic N-oxidation of N-ethyl-N-methylaniline (EMA) was investigated in vitro following incubation of the compound (1mM) with fortified hepatic microsomal preparations of both male Wistar rats and New Zealand White (NZW) rabbits. The major metabolites in both species were found to be N-ethylaniline, N-methylaniline and EMA N-oxide. Chromatographic resolution of the N-oxide enantiomers was achieved using a Chiralcel OD stationary-phase with a mobile-phase of hexane:ethanol (98:2, v/v). Examination of the enantiomeric composition of the N-oxide metabolites indicated a predominance of the (-)-(S)-N-oxide from both species with enantiomeric excesses of 52 +/- 2.5% and 65 +/- 2.1% (n = 3) in rat and rabbit tissue respectively. These preliminary observations indicate that the N-oxidation of EMA shows product stereoselectivity, the extent of which varies between species.

Metabolism of temelastine (SK&F 93944) in hepatocytes from rat, dog, cynomolgus monkey and man.

A species comparison of the metabolic pathways of temelastine has been made using hepatocyte preparations from rat, dog, cynomolgus monkey, and man. Metabolites and unchanged temelastine were separated by HPLC and were compared with authentic standards by retention. The characteristic UV spectra of SK&F 93944 and its metabolites aided in the preliminary identification of metabolites in hepatocyte incubates, subsequently confirmed by liquid chromatography/mass spectrometry (LC/MS). The metabolic profile of temelastine is complex, both in vivo and in vitro, but all of the metabolites identified unambiguously from in vivo studies have also been demonstrated in vitro. Moreover, the time-dependent nature of the metabolic profile has been investigated in rat hepatocytes. Marked differences in the rate of production, extent of accumulation, and distribution between cells and culture medium have been observed for specific metabolites. Species differences in the metabolism of temelastine by rat, dog, cynomolgus monkey, and human hepatocytes have been observed. In particular, SK&F 94224 (a hydroxylated metabolite of temelastine) was not detected in human hepatocyte incubations at appreciable concentrations, but was present in varying amounts in the other species and especially in incubations from dog hepatocytes. Temelastine N-glucuronide was not detected in the rat hepatocyte system but was present to a modest or significant extent in hepatocyte incubations from dog, cynomolgus monkey, and man.

The antipyrine breath test in the rat: a pharmacokinetic model.

Breath tests have been widely advocated for use as non-invasive probes of mixed function oxidase activity in vivo. A catenary sequence of events begins with demethylation and results in the exhalation of 14CO2. Intermediates in this chain include formaldehyde and formate. In this current study [14C]-antipyrine, [14C]-formaldehyde and [14C]-formate have been administered to rats. The data from these one carbon intermediates lead to the conclusion that demethylation is not the rate-limiting step in the antipyrine breath test in the rat. The resultant 14CO2 exhalation rate time profiles have been used to derive a compartmental pharmacokinetic model for the antipyrine breath test in the rat. The simplest catenary model (Antipyrine----formaldehyde----formate----CO2) did not adequately describe the observed data. A compartment in equilibrium with the central compartment for formate was needed to characterize fully the observed data. The derived compartmental model was able to predict qualitatively the effects of phenobarbitone induction on the antipyrine breath test. The quantitative agreement between the model prediction and the observed data could be improved by incorporating the changes in one carbon metabolism produced by phenobarbitone.

The interaction of omeprazole with rat liver cytochrome P450-mediated monooxygenase reactions in vitro and in vivo.

The effect of omeprazole on cytochrome P450-mediated monooxygenase reactions was assessed in rat liver S9 utilising ethylmorphine-N-demethylase (EM) and ethoxycoumarin-O-deethylase (ECOD) activities. The inhibition of EM by omeprazole was judged to be predominantly reversible in mechanism. The average Ki for omeprazole was 40 +/- 27 microM with EM and 76 +/- 6 microM with ECOD in four separate rats. In preparations of rat hepatocytes the intrinsic clearance of diazepam was decreased substantially by 50 microM omeprazole (average inhibition 73%). In comparison 50 microM cimetidine inhibited the intrinsic clearance of diazepam by 50%. The relationship between these two in vitro models for drug interactions is discussed in the context of previously published drug inhibition data. Moreover, repeated administration of omeprazole to adult male rats (500 mg.kg-1, 14 days, p.o.) resulted in statistical increases in liver weight, cytochrome P450 and ECOD activity. Thus omeprazole interacts with the mixed function oxidase system in vitro and in vivo.

Passage of paracetamol into breast milk and its subsequent metabolism by the neonate.

1 Paracetamol was administered to nursing mothers. The drug passed rapidly into milk and the milk:plasma concentration ratio was approximately unity. 2 The estimated maximum dose to the neonate was 1.85% of the weight-adjusted maternal oral dose of paracetamol 1.0 g. Recovery of paracetamol was greater from the breast from which samples were taken frequently than from the breast which was sampled only once. 3 Paracetamol, its glucuronide, sulphate, cysteine and mercapturate conjugates were found in the urine of the neonates although only the parent drug was detected in breast milk. 4 The neonates excreted significantly greater proportions of unchanged paracetamol (P less than 0.01) and significantly lesser proportions of paracetamol sulphate (P less than 0.001) than did healthy volunteers aged 11-80 years who received a therapeutic dose of paracetamol. 5 The findings are compatible with a deficiency of sulphate conjugation by the neonate.

Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man.

Diazepam metabolism has been investigated in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man. The metabolite profile obtained by HPLC analysis of the culture medium indicated that substantial differences exist corresponding to known species differences in the metabolite profile of diazepam in vivo. These differences were attributed to a combination of the rate at which a metabolite was formed and the rate at which it is removed from the medium by further metabolism. The intrinsic clearance of nordiazepam in hepatocytes from each of the species exhibited the most marked species variation (rat much greater than guinea pig greater than rabbit greater than human greater than dog). Species that exhibited a high intrinsic clearance for nordiazepam were also those species that exhibited significant hydroxylation at the 4'-site of the molecule. The disappearance of diazepam was rapid in rat, dog, and guinea pig hepatocytes, but slow in human hepatocytes. Moreover, rat and human hepatocytes exhibited different saturability of diazepam clearance with respect to diazepam concentration accounting, at least in part, for the different rates of diazepam metabolism in the different species. These results support the value of hepatocytes in drug metabolism studies and especially in studies of species differences in metabolism.

Primary cultures of adult rat hepatocytes--a model for the toxicity of histamine H2-receptor antagonists.

Oxmetidine, a potent histamine H2-receptor antagonist, is cytotoxic to primary cultures of adult rat hepatocytes. The criteria of cellular injury included leakage of cytoplasmic enzymes into the culture medium, inhibition of protein synthesis and measurement of oxygen consumption by freshly isolated hepatocytes. These parameters were correlated with morphological changes in the cells as judged by inverted phase-contrast microscopy. In contrast, two other histamine H2-receptor antagonists, cimetidine and ranitidine, caused only minor changes in these parameters of cytotoxicity. The extent of injury observed with oxmetidine was both time and concentration dependent and was similar in hepatocytes maintained in culture for 2, 24 or 48 h. Prior treatment of rats with phenobarbital or beta-naphthoflavone did not influence oxmetidine-induced cytotoxicity. Inhibitors of cytochrome P-450-mediated monooxygenase activity had little effect on oxmetidine-induced injury with the exception of metyrapone, which was shown to inhibit the observed cytotoxicity by a mechanism other than inhibition of monooxygenase activity. In contrast, the injury could be potentiated by L-ethionine, an antimetabolite which reduces cellular ATP levels, suggesting that oxmetidine induces cytotoxic effects as a consequence of an interaction with intermediary energy metabolism.

The effect of cimetidine and ranitidine on paracetamol glucuronidation and sulphation in cultured rat hepatocytes.

Cimetidine and ranitidine have been investigated for their ability to inhibit conjugation reactions in cultures of rat hepatocytes. Neither compound had any appreciable effect on rates of paracetamol sulphation. However, both cimetidine and ranitidine inhibited the glucuronidation of paracetamol in a dose-dependent manner. No adverse effects on cellular viability were noted utilizing enzyme leakage (lactic dehydrogenase) or protein synthesis measurements. The kinetics of inhibition by ranitidine were studied in more detail. At 0.25 mM ranitidine, the inhibition appeared to be purely competitive. However, at higher concentrations decreases in Vappmax were noted suggesting a more complex mechanism of inhibition. The relevance to inhibition in vivo by cimetidine and ranitidine and possible interactions between paracetamol and these histamine H2-receptor antagonists are discussed.

Comparison of the new miniature Wright peak flow meter with the standard Wright peak flow meter.

Preproduction and current models of the miniature Wright peak flow meter have been compared with the standard Wright peak flow meter on normal and abnormal subjects. Early problems in production appear to have been overcome, and the current model agrees to within 3% with the standard peak flow meter, which is as close as the agreement between two standard instruments. The new mini-meter may be enclosed in a case, making direct comparisons with other instruments possible.